DESCRIPTION: (Investigator's abstract) Investigation of the mechanism of activation of human plasminogen by streptokinase is proposed with the long-term goal of determining the molecular mechanism by which SK activates the human fibrinolytic system, which is the basis for its use as a thrombolytic drug for the treatment of cardiovascular diseases. In the current hypotheses, activation of Pg by SK involves two linked modes of interaction. Pg activation is initiated by the unique function of SK to induce proteinase activity in Pg nonproteolytically, through specific binding and reversible induction of a conformational change that activates the catalytic site of the zymogen. Conformational activation of Pg in this mode of interaction is hypothesized to act as the triggering event in the proteolytic activation of Pg to plasmin (Pm). Proteolytic cleavage of Pg is thought to be catalyzed initially by conformationally activated Pg in SK-Pg complexes and propagated subsequently by SK-Pm complexes formed by high affinity binding of Pm. Conformational activation of Pg by SK and the ensuing proteolytic activation process are modulated by differences in the interactions of SK with Pg and its proteolytic activation products, and the involvement of lysine binding sites on these species in the interactions. To induce proteolytic activation of Pg, SK redirects drastically the substrate specificity of Pm from that of a proteinase which does not activate Pg to a specific Pg activator. The origin of the change in substrate specificity is hypothesized to involve a conformational change accompanying SK binding to Pm which affects the active site to enhance specificity for the cleavage-site sequence in Pg. Moreover, the acquisition of specificity for Pg activation is postulated to involve a second mode of Pg interaction at a site expressed on SK-Pm and SK-Pg complexes that functions as a specific, protein-substrate-recognition exosite to facilitate binding and cleavage of Pg. Novel derivatives of Pg that are specifically labeled with fluorescence probes at the catalytic site of the zymogen have been developed which provide new tools for the proposed evaluation of the mechanism of Pg activation, by the use of fluorescence spectroscopy, protein chemistry, and enzyme kinetic techniques. Specific aims are: (1) Quantitative characterization of SK-induced conformational activation of Pg under reversible, equilibrium conditions. (2) Determination of the origin of the SK-induced change in substrate specificity of Pm and the mechanism of SK-induced proteolytic activation of Pg. (3) Definition of the reaction mechanism of conformational activation of Pg by SK. (4) Identification of structural sites of SK-Pg interactions and determination of their functional roles in the activation mechanism. The results of the proposed investigation are expected to have significance in elucidating fundamental molecular events in the action of SK as a thrombolytic drug, which may provide the basis for improving SK therapy and enable the design of more clinically effective thrombolytic agents.